T cells often lose their ability to continue making robust responses, when stimulated for a long time by a persistent antigen. This is a significant problem, because established tumors and chronic infections can also evade protective immune responses by inducing a similar state of tolerance in antigen-specific T cells - known as exhaustion or anergy. This project identifies novel regulatory molecules that help maintain such a tolerance. The molecular mechanics of tolerance is understood to include the expression of inhibitory molecules such as PD1, LAG3, CTLA4 etc. which dampen different intracellular activating signals in T cells. Accordingly, recent clinical studies have successfully used antibodies and inhibitors against such negative regulators to augment T cell responses to tumors and chronic viral infections. This proposal evaluates the hypothesis that two newly identified regulators - PEAR1 and EndoD1 - cooperatively inhibit the responsiveness of CD4+ T cells, contributing to T cell tolerance. It is projected that inhibiting these molecules in T cellscan improve the efficacy of immunotherapy, e.g. against tumors - potentially even in contexts where targeting known regulators alone is insufficient. These hypotheses will be evaluated using three independent and complimentary aims. 1. Determine how these novel regulators individually and synergistically affect T cell activation in vivo in the context of autoimmunity and graft rejection by using a T cell-specific knockdown approach. In addition to preliminary data in an arthritis model, the impact of the knockdowns on T cells in a dermatitis model will be evaluated by enumerating activation marker expression, cytokine production as well as immunopathology. 2. Elucidate the biochemical targets of these regulators in tolerant T cells, with a view to developing a rational strategy to identify permutations of pathways that can be most efficiently targeted for immunotherapy. A combination of molecular analyses and computational modeling will be used for this purpose. 3. Define a strategy to target these regulators during adoptive T cell therapy for tumors by knocking-down their expression in tumor-specific transgenic T cells as well as native tumor-infiltrating lymphocytes. It is expected that these experiments will, for the first time, define the role of T cell expressed PEAR1 and EndoD1 as inhibitors of T cell effector responses. This is significant because it offers new targets for therapeutically (a) enhancing the function of antigen-specific T cells in the context of immunity to tumors and chronic infections or (b) tolerising T cells during autoimmunity and graft rejection. Furthermore, the proposed studies on the cooperativity of these molecules with other known negative regulators of T cell activation, such as PD1, CTLA4, cbl-b and LAG3, will generate data required to rapidly integrate these targets with ongoing clinical efforts. A rational strategy to guide the design of such combinatoria treatments, using biochemical data and computational models, is therefore also included in the proposal.